PROJECT SUMMARY Cytosine DNA methylation is a post-replicative epigenetic modification that regulates the organization and expression of the genetic information. Deficiencies in DNA methylation associate with numerous diseases, most notably genomic imprinting disorders, immunodeficiencies, and cancers. In the vertebrates, cytosine methylation happens in the CpG context. DNA replication produces a short-lived hemimethylated DNA intermediate. Due to its fleeting nature and the lack of adequate tools, DNA hemimethylation remains underexplored. Intriguingly, recent studies revealed fascinating examples of persistent DNA hemimethylation. These studies suggest a broader spectrum of functional states of DNA methylation. To begin to address this gap in our understanding of this potentially crucial element of the epigenetic regulation of the genome, we propose to adapt a SET- and RING Finger-Associated (SRA) domain of UHRF1 to generate a DNA hemimethylation probe for in vitro and in vivo studies. The compact SRA domain is self-sufficient to bind hemimethylated DNA with high specificity. To accomplish the overarching goal of this proposal, we will first produce recombinant SRA domain proteins and develop methods for the isolation and visualization of hemimethylated DNA in complex genomes in vitro. To study DNA hemimethylation in vivo, we will express a fusion protein comprised of the SRA domain and mScarlet fluorescent protein in mouse embryos, embryonic stem cells, and transgenic mice. We will use live imagining of cells with hemimethylated DNA to validate chromatin localization patterns and dynamics of the SRA-mScarlet fusion protein. This research will enhance our understanding of the epigenetic regulation of the genome in development and disease.